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1 University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Theses and Dissertations in Animal Science Animal Science Department EFFECTS OF PUBERTAL STATUS AND NUMBER OF ESTROUS CYCLES PRIOR TO BREEDING IN BEEF HEIFERS, AVERAGE DAILY GAIN ON REPRODUCTIVE PERFORMANCE AND COMPARISON OF TWO FIXED TIME AI ESTRUS SYNCHRONIZATION PROTOCOLS Rebecca Vraspir University of Nebraska-Lincoln, bvraspir15@gmail.com Follow this and additional works at: Part of the Animal Sciences Commons Vraspir, Rebecca, "EFFECTS OF PUBERTAL STATUS AND NUMBER OF ESTROUS CYCLES PRIOR TO BREEDING IN BEEF HEIFERS, AVERAGE DAILY GAIN ON REPRODUCTIVE PERFORMANCE AND COMPARISON OF TWO FIXED TIME AI ESTRUS SYNCHRONIZATION PROTOCOLS" (2014). Theses and Dissertations in Animal Science This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Theses and Dissertations in Animal Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln.

2 EFFECTS OF PUBERTAL STATUS AND NUMBER OF ESTROUS CYCLES PRIOR TO BREEDING IN BEEF HEIFERS, AVERAGE DAILY GAIN ON REPRODUCTIVE PERFORMANCE AND COMPARISON OF TWO FIXED TIME AI ESTRUS SYNCHRONIZATION PROTOCOLS by Rebecca A.Vraspir A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science Major: Animal Science Under the Supervision of Professors Richard N. Funston and Jennifer R. Wood Lincoln, NE August, 2014

3 EFFECTS OF PUBERTAL STATUS AND NUMBER OF ESTROUS CYCLES PRIOR TO BREEDING IN BEEF HEIFERS, AVERAGE DAILY GAIN ON REPRODUCTIVE PERFORMANCE AND COMPARISON OF TWO FIXED TIME AI ESTRUS SYNCHRONIZATION PROTOCOLS Rebecca A. Vraspir, M.S. University of Nebraska, 2014 Advisors: Richard N. Funston and Jennifer R. Wood Reproductive success is the most important factor in beef cattle production and is affected by timing of pubertal onset in heifers, and reproductive biotechnologies utilized. Three studies were conducted to evaluate the effects of pubertal status, ADG and two fixed time AI protocols on reproductive success in beef heifers. In the first study, Heifers that were pubertal prior to breeding had a greater AI and overall pregnancy rate, produced more calves born within the first 21 day of the calving season, and weaned older, heavier calves than non-pubertal heifers. Additionally, number of estrous cycles prior to breeding tended to influence pregnancy rates and heifers that had 2 estrous cycles, prior to the first breeding season, had a greater second season pregnancy rate than those heifers that had 0 or 1 estrous cycle prior to the first breeding season. In the second study, as ADG increased leading up to breeding the odds of attaining puberty increased for GSL heifers. For NP heifers, as ADG increased the odds

4 of puberty attainment decreased. Odds of pregnancy were affected by body weight gain and pubertal status interaction; however, pubertal status had the greatest influence on increasing the odds of pregnancy. In the third study, two progestin-based fixed time AI protocols, MGA and 14-d CIDR, were compared to evaluate pregnancy rates. Fixed time AI pregnancy rate and final pregnancy rate was similar between MGA and 14-d CIDR. An economic analysis was performed and determined the synchronizing heifers with MGA was more costeffective in this study. In summary, if a heifer attains puberty prior to the breeding season acceptable pregnancy rates can be achieved regardless of the number of estrous cycles experienced prior to breeding. However, second season pregnancy rates may be affected. Additionally, MGA and 14-d CIDR produce similar and acceptable fixed time AI pregnancy rates.

5 i Table of Contents Chapter I: Literature Review Introduction...1 Estrous Cycle...2 Puberty...5 Reproductive tract development...6 Preweaning management...8 Postweaning management...9 Estrous Synchronization...11 MGA...12 CIDR...17 Conclusions/Objectives...19 Literature Cited...21 Chapter II: Effect of pubertal status and number of estrous cycles prior to the breeding season on pregnancy rate in beef heifers...25 Abstract...25 Introduction...26 Materials and Methods...27 Results and Discussion...29 Literature Cited...33 Tables...35 Chapter III: Effect of average daily gain (ADG) on pubertal status and pregnancy in beef heifers...38 Abstract...38 Introduction...39 Materials and Methods...41 Results and Discussion...43 Literature Cited...49 Tables...51 Chapter IV: Comparison of melengestrol acetate and controlled internal drug release long-term progestin-based synchronization protocols on fixed-time AI pregnancy rate in beef heifers...61 Abstract...61 Introduction...62 Materials and Methods...63 Results and Discussion...66 Literature Cited...69 Tables...72

6 ii List of Tables and Figures Chapter II: Effect of pubertal status and number of estrous cycles prior to the breeding season on pregnancy rate in beef heifers Table 1. Birth date, BW, pregnancy rate, and first calf characteristics of heifers classified by pubertal status prior to breeding. (Exp. 1)...35 Table 2. Birth date, BW, ADG, pregnancy rate, and first calf characteristics of heifers classified by pubertal status prior to breeding. (Exp. 2)...35 Table 3. Birth date, BW, ADG, pregnancy rate, and first calf characteristics of heifers classified by number of estrous cycles prior to breeding. (Exp. 3)...36 Chapter III: Effect of average daily gain (ADG) on pubertal status and pregnancy in beef heifers Table 1. Effect of birth to weaning ADG on pubertal status (GSL) Table 2. Effect of weaning to breeding ADG on pubertal status (GSL) Table 3. Effect of pubertal status on pregnancy rate (GSL) Table 4. Effect of weaning to breeding on pubertal status (NP) Table 5. The effect of one month prior to breeding ADG on pubertal status (NP) Table 6. The effect of AI ultrasound to final pregnancy ultrasound on final pregnancy rates (NP) Figure 1. Interaction of pubertal status and breeding to pregnancy ultrasound on pregnancy rates (GSL) Figure 2. The effect of ADG from AI to AI pregnancy ultrasound on AI pregnancy rates (NP) Figure 3. The interaction of ADG from AI to AI pregnancy ultrasound on final pregnancy rates (NP) Figure 4. The interaction of ADG from AI to final pregnancy ultrasound on final pregnancy rates (NP) Chapter IV: Comparison of melengestrol acetate and controlled internal drug release long-term progestin-based synchronization protocols on fixed-time AI pregnancy rate in beef heifers Table 1. Composition and nutrient analysis of drylot diet fed to heifers...72 Table 2. Reproductive measurements prior to treatment and effect of controlled internal drug release (14-d CIDR) and melengestrol acetate (MGA) synchronization systems on pregnancy rates...73

7 iii Table 3. Cost comparison of controlled internal drug release (14-d CIDR) and melengestrol acetate (MGA) synchronization protocols...74 Figure 1. Treatment schedule for heifers assigned to melengestrol acetate or 14-d controlled internal drug release...76

8 1 Chapter I: LITERATURE REVIEW Introduction Beef cow-calf operations productivity and profitability is dependent on the development of beef heifers as replacement breeding animals for the cow herd. Profitability is directly influenced by a cow s reproductive lifespan and her ability to wean a marketable calf each year. In order for replacement heifers to be generated with these characteristics, producers must produce a pregnant heifer early in the first breeding season without excessive development costs and manage the heifer to return to estrus and become pregnant early in the following breeding seasons. This is important as it takes 3 to 5 weaned calves to recover the development costs for a replacement heifer. Multiple factors contribute to optimal heifer reproductive performance, but first and foremost is achievement of puberty prior to the first breeding season. Development of heifers to have 1 to 3 estrous cycles prior to their first breeding season is the industry standard, as heifers are more likely to become pregnant when they have had 3 estrous cycles prior to breeding compared to having only one estrous cycle (Byerley et al., 1987). The management tools that impact age at puberty are genetics and nutrition. However, selection for accelerated genetics for reproductive traits may take several generations to see results, whereas proper nutrition can produce more immediate results in attainment of puberty and fertility. Research has shown that heifers of similar breed composition can reach puberty several months apart when developed on different diets (Wiltbank et al., 1969; Short and Bellows, 1971). However, the differences in age at puberty due to differing nutritional

9 2 regimes comes with great financial impact, as 60 to 70% of heifer development costs are attributed to feed. Therefore, the cost of an earlier age at puberty needs to be weighed against the profits to be made by increased pregnancy rates and heavier weaning weights. In addition, there are many technologies that can be implemented in a management system to help induce puberty, have more heifers achieve pregnancy early in the breeding season, and inseminate heifers with semen from proven sires for calving ease or low birth weights. Two of these reproductive biotechnologies are estrus synchronization and AI. They have been available for more than 30 years, however producers have been slow to adopt them. Perhaps this is due to labor intensity and costs associated with estrus synchronization pharmaceuticals and semen for AI; however, labor intensity is dramatically decreased with the use of fixed-time AI (FTAI), while still producing acceptable pregnancy rates. Again, the use of these technologies can hasten puberty onset in peri-pubertal heifers, concentrate the breeding period and calving period, reduce incidences of dystocia if high accuracy, low birth weight bulls are utilized. Estrous Cycle The cyclical pattern of ovarian activity that facilitates sexual receptivity, allowing for mating, and establishment of pregnancy is called an estrous cycle (Forde et al., 2011). Cattle have an estrous cycle that consists of 2 phases: the luteal phase and the follicular phase. The luteal phase is the period following ovulation through the formation and lifespan of the corpus luteum. The follicular phase is the period from the regression of the corpus luteum to ovulation (Forde et al., 2011). These phases can also be broken down

10 3 into 4 stages, proestrus and estrus, which occur during the follicular phase, and metestrus and diestrus make up the luteal phase. The average length of the estrous cycle in cattle is 21 d; however, the length is determined by the number of follicular waves, which varies between individual cows. Cattle most commonly have 2 or 3 follicular waves with a new wave starting every 6 to 8 d, therefore the estrous cycle can range from 18 to 24 d (Figure 1). Figure 1. The illustration above depicts the follicular waves that occur throughout the bovine estrous cycle. The yellow follicles are healthy growing follicles, whereas the red follicles are atretic. Additionally, patterns of follicle stimulating hormone (FSH; blue), luteinizing hormone (LH; green) and progesterone (P4; orange) are depicted. Ovulation is induced by a surge of LH and FSH. (Adapted from Forde et al., 2011). The estrous cycle is under endocrine regulation of the hypothalamic-pituitarygonadal axis (HPG) and functions under positive and negative feedback systems, which differs from the follicular phase to the luteal phase.

11 4 Follicular Phase Proestrus is the stage of the estrous cycle that leads to estrus, where ovulation occurs. During proestrus, progesterone concentration decreases. Lower progesterone concentration decreases the negative feedback progesterone plays on the hypothalamus, increasing GnRH release; as well as the anterior pituitary, increasing secretion of gonadotropins, LH and FSH. The increase in GnRH allows for stimulation of gonadotropin release, allowing for follicular growth, thus increased concentration of estrogen (Figure 2). According to Roche (1996), when serum progesterone levels are basal and LH pulses occur every 40 to 70 minutes. Estrus is the stage of the estrous cycle that follows proestrus, in which estrogen concentrations are continuing to rise until a threshold concentration or peak is reached. The peak concentration of estrogen (positive feedback to the hypothalamus) causes a large quantity of GnRH to be released from the surge center, which stimulate the anterior lobe of the pituitary to secrete the preovulatory surge of LH (Sunderland et al., 1994). The preovulatory surge of LH is at least 10 times greater than the tonic pulses of LH. The LH surge causes ovulation of the dominant follicle. Ovulation occurs approximately 10 to 14 h after the observed standing estrus.

12 5 Figure 2. The figure above illustrates the hypothalamic-pituitary-gonadal (HPG) axis during the follicular phase of the estrous cycle. The positive feedback mechanism of estrogen (E 2 ) on the surge center of the hypothalamus is depicted, stimulating a surge release of gonadotropin releasing hormone (GnRH), thus stimulating the pre-ovulatory surge of lutenizing hormone (LH) from the anterior pituitary. (Adapted from Senger, 2012). Luteal Phase Metestrus, the stage of the estrous cycle that follows estrus, is the beginning of the luteal phase. Metestrus occurs during the first 5 d of the estrous cycle (ovulation being d 0) and formation of the corpus luteum (CL) occurs during this stage. Once the follicle ruptures during ovulation, blood vessels within the follicular wall also rupture, giving the

13 6 area on the ovary where the follicle was located a bloody appearance, known as a corpus hemmorhagicum. When the ruptured follicle collapses, it causes many folds, where the cells of the theca interna and granulosa cells begin to mix, forming a gland that consists of connective tissue, theca cells and granulosa cells (Senger, 2012). Theca cells and granulosa cells then undergo a dramatic transformation in to luteal tissue called lutenization, which is governed by LH. This structure becomes the CL and starts to produce progesterone. Progesterone is the hormone responsible for preparing the uterus for implantation and maintaining pregnancy. During diestrus, the CL continues to grow for the first few days, then reaches its maximum growth and progesterone secretion peaks and remains constant for approximately 10 d (Figure 3). Progesterone being secreted by the CL exhibits a negative feedback on GnRH secretion from the hypothalamus. During the luteal phase, estrogen also exhibits negative feedback on the hypothalamus and anterior pituitary. The negative feedback that occurs during the luteal phase prevents ovulation from occurring when the HPG is under control of progesterone. If recognition of a pregnancy (interferon-τ) is not detected by d 18 of the estrous cycle, prostaglandin F 2α (PGF 2α ) is produced by the uterus to regress the CL and decrease progesterone concentrations produced (luteolysis), which occurs the last 2 days of diestrus. Luteolysis is the irreversible degradation of the CL, thus causing a decrease in progesterone concentration and removing the negative feedback.

14 7 Figure 3. The figure above illustrates the hypothalamic-pituitary-gonadal (HPG) axis during the luteal phase of the estrous cycle. The negative feedback of progesterone (produced by the corpus luteum on the ovary) on hypothalamus and anterior pituitary are depicted. (Adapted from Senger, 2012). During the luteal phase, recurrent waves of follicle development continue; however, the negative effect of the high level of progesterone does not allow LH to be secreted at a frequency high enough to cause ovulation, therefore those follicles become atretic (Rahe et al., 1980).

15 8 Puberty Normal estrous cycles, with ovulation of a dominant follicle and luteal phase of 15 to 17 d in length, do not occur in heifers until after they have reached puberty. Puberty is defined as the first ovulatory estrus followed by a luteal phase of normal length (15 to 17 d) and is the first opportunity for a heifer to conceive (Atkins et al., 2013). Estrus and ovulation can occur independently of each other in peri-pubertal heifers. Therefore, nonpubertal estrus or ovulation without estrus should not be confused with puberty, where ovulation is accompanied by behavioral estrus. The part of the endocrine system that regulates the onset of puberty is the HPG. Prior to puberty, GnRH neurons within the hypothalamus are highly sensitive to a strong negative inhibition by estradiol; however, the negative effect of estradiol decreases as heifers mature and approach puberty. Changes in GnRH neuron sensitivity to estradiol initiates puberty, allowing for GnRH to be released at the appropriate frequency and quantity to stimulate gonadotropin secretion. Gonadotropins, LH and FSH, are produced and released by the anterior pituitary. Follicle stimulating hormone causes follicular growth, an increase in estradiol secretions as follicle diameter increases, and an increase in LH pulse frequency, leading to an LH surge and ovulation (Kinder et al., 1995; Day and Anderson, 1998). Throughout development of the HPG in heifers, the amount of GnRH in the hypothalamus does not change, only the sensitivity of GnRH neurons changes (Kinder et al., 1995). No morphological changes have been observed in GnRH neurons, however

16 9 changes in estrogen receptor and kisspeptin within the hypothalamus have been identified as major players in puberty initiation (Anderson and Day, 1996). Within the hypothalamus GnRH neurons do not contain estrogen receptors (ER); however, ER are located in many other areas within the hypothalamus, such as medial preoptic area (MPOA), anterior hypothalamus (AH), ventrolateral septum, bed nucleus of stria terminalis, ventromedial hypothalamus (VMH), and the arcuate nucleus (ARC; Day and Anderson, 1998; Atkins et al., 2013). Neurons that are ER-positive decrease in the AH and medial basal hypothalamus (MBH) as puberty approaches, which has been reported to be negatively correlated with LH pulse frequency (Day et al., 1987). The exact mechanisms that stimulate the change in sensitivity of GnRH neurons to estradiol have not been elucidated, but metabolic signals seem to be involved. The age at which beef females reach puberty can be affected by many factors, some of particular interest are weight, plane of nutrition, and ADG prior to and following weaning. Reproductive tract development Prenatal development Reproductive organs start to develop well before birth in beef heifers, with ovary development occurring by d 50 to 60 of gestation and primordial follicles identified on fetal bovine ovaries by d 74 to 80 of gestation (Hubbert et al., 1972; Tanaka et al., 2001; Nilsson and Skinner, 2009). Concentrations of FSH and estradiol have been detected around mid-gestation in the bovine fetus, which continue to increase throughout the duration of gestation (Tanaka et al., 2001). The maturation of reproductive tissues and the endocrine axis continues after birth.

17 10 Postnatal development Reproductive development,which was initiated in-utero, continues through the peri-pubertal stage in beef heifers, with wave-like patterns of follicular development being observed as early as 2 wk of age in heifer calves (Gasser, 2013). Honaramooz et al. (2004) evaluated the reproductive tissues of Hereford heifers every 2 wk from 2 to 60 wk after birth via transrectal ultrasonography. From this study, important phases of growth were identified in beef heifers. Ovarian dimensions increased from 2 to 14 wk and again after 34 wk. The size of the largest ovarian follicles increased from 8 to 14 wk, 38 to 42 wk, and 52 to 60 wk. The number of follicles 3 mm in diameter tended to increase from 6 to 14 wk and significantly increased from 6 to 60 wk of age. The first ovulation occurred, on average, at 63.7 wk of age. The heifers in this study were gaining BW at an average of approximately 2 lb/d throughout this study with heifer BW at puberty averaging 883 lb. This study gives insight on how nutrition during some of these time points could influence reproductive tract development, and potentially times of development to put more emphasis on nutrient intake. It is known that reproductive development starts inutero, so attention should be paid to dam nutrition during gestation. Additionally, from the birth to 14 wk and after 34 wk ovarian development occurs, growth in diameter and number of follicles, meaning these may be times of development to put more research focus on to see how nutrition during these periods affect age at puberty and number of antral follicles.

18 11 Pre-weaning management Many studies have provided evidence that diet during development can account for some of the physiological changes necessary for the attainment of puberty (Frisch, 1984). A hormone called leptin, may be one of the main players in effect that diet has on puberty initiation. Leptin is a hormone that signals nutritional status to the HPG; its expression and secretion have been correlated with body fat mass (Zieba et al., 2005). Heifers of similar breed composition can reach puberty several months apart when fed different diets (Wiltbank et al., 1969; Short and Bellows, 1971). In a study looking at BW and age at puberty in Hereford heifers done by Arije and Wiltbank (1974) found heifers that had a greater growth rate from birth to weaning, heavier weaning weights and reached puberty earlier, at a heavier BW than their herd mates with a slower growth rate prior to weaning. Additionally, they found those heifers that grew more rapidly after weaning tended to be heavier at puberty, but not necessarily younger. Wiltbank et al. (1966) found that regardless of overwinter feeding treatment, preweaning ADG had a significant effect on age at puberty, with a 0.1 kg increase in ADG leading to an 18.7 d decrease in age at puberty. When evaluating the effect of postweaning ADG on age at puberty, it only had a significant effect if heifers were on a low level of nutrition over winter. Wiltbank et al. (1966) determined that age at puberty was more consistently affected by preweaning BW gain versus post-weaning BW gain. Gasser (2013) reviewed multiple studies that focused on the peripubertal period in heifers. These studies included early weaning and feeding a high concentrate diet to heifers. These heifers had a substantially reduced age at puberty, increased ovarian

19 12 maturation, and increased estradiol concentrations during follicular waves. This research provides evidence that nutritional influence during the preweaning can greatly influence timing of puberty, and thus reproductive performance. Well-controlled studies on pre-weaning nutrition are limited; many studies have estimated pre-weaning growth rate and weaning weight (Hall, 2013). The dams milking ability may be the largest factor contributing to the importance of pre-weaning growth and its role in fertility. Maternal milk production influences calf weaning weight, which research has shown plays a role in puberty (Corah et al., 1975). Additionally, Gasser et al. (2006) have shown nutrient status within the first 2 to 3 months of age impacts the onset of puberty. Post-weaning management For approximately 20 years, industry standards have been to develop replacement heifers to 65% of their mature BW in order to ensure attainment of puberty prior to the breeding season (Patterson et al., 1992). Many studies were conducted to determine whether puberty was controlled by age or BW, and studies have shown that rate and timing of BW gain can influence age at puberty (Wiltbank et al., 1966; Arije and Wiltbank, 1974; Lynch et al., 1997). However, research in the past decade has challenged the 65% of mature BW rule. As feed costs have increased, it has been important to evaluate how much nutrition is actually required to maintain heifer reproductive performance. Currently heifers are developed to reach puberty by 12 to 15 months of age so they can conceive and calve as a 2-yr old. Byerley et al., (1987) discovered the fertility to

20 13 the first estrus was 21 percentage points less than heifers inseminated on the third estrus. Therefore, it has been an industry standard to develop heifers to attain puberty 1 to 3 months prior to the breeding season. With the increased cost of feedstuffs it is important that emphasis is put on nutrition during critical growth periods to allow heifers to attain puberty in time to have multiple estrous cycles prior to the breeding season. It is still in question if pre-weaning or post-weaning nutrition has a greater influence on heifer reproductive performance. Roberts et al. (2009) nutrient restricted heifers for 140 d after weaning, by doing this the proportion of heifers that attained puberty by 14 months was decreased; however, by the end of the breeding season pregnancy rates were similar between the restricted heifers and the control heifers. In another study done by Funston and Deutscher (2004), heifers were developed to either 53% or 58% of their mature BW with no significant differences in calving interval, calving date, or pregnancy rate through the third breeding season. (Short and Bellows, 1971) fed heifers to gain either 0.45 kg/d or 0.68 kg/d. Heifers that gained.68 kg/d achieved puberty approximately one month earlier than those that gained 0.45 kg/d. One postweaning study done by (Funston and Larson, 2011) looked at 2 postweaning heifer development systems, a traditional drylot development system (DL) and an extensive grazing system (EXT) utilizing crop residue and winter range. The heifers in the DL system were heavier throughout the breeding season (387 vs. 336 kg) and a greater proportion of the DL heifers were pubertal before the breeding season (88 vs. 48%); however, by the end of the breeding season pregnancy rates were similar between DL and EXT heifers.

21 14 Earlier studies suggest postweaning gain has a greater influence on heifer age at puberty and pregnancy rates, than what has been shown in more recent research. Genetic advancements that have taken place over the past couple decades, such as selection for increased scrotal circumference that is negatively correlated with age of puberty in female progeny, may be playing a large part in the changes seen over time in attainment of puberty and heifer fertility. Estrus Synchronization Estrus synchronization is one of the most important and advantageous reproductive techniques that has been available for use by cattle producers for several decades. The advantages to using estrus synchronization are that estrus occurs during a predicted time range which allows for AI, embryo transfer, or other reproductive techniques to be utilized. This provides the opportunity for more females to conceive earlier in the breeding season, thus leading to a more concentrated calving season and calves being more uniform in age and weight at weaning. Research has shown that calves born within the first 21 d of the calving season are older and heavier at weaning and have greater lifetime productivity than their contemporaries born later in the calving period (Lesmeister et al., 1973; Funston et al., 2012). By utilizing estrus synchronization, with or without AI, females have a greater opportunity to become pregnant early in the breeding season, thus will calve earlier in the calving season. Estrus synchronization was developed in 6 phases, as researchers began to understand the mechanisms that controlled the estrous cycle in cattle. The first discovery was that progesterone inhibited ovulation (Ulberg et al., 1951) and understanding the

22 15 maturation of pre-ovulatory follicles (Hansel et al., 1961; Lamond, 1964). Therefore, the initial protocols of estrus synchronization development centered on control of the luteal phase, as the follicular waves had yet to be recognized. The Progesterone Phase, was the first phase, where exogenous progesterone was administered to cattle to prolong an existing or establish an artificial luteal phase. The second phase, Progesterone-Estrogen, used estrogens and gonadotropins to manipulate the estrous cycle. The third phase, PG phase, came about in 1972 when prostaglandin and its analogs were found to be luteolytic in cattle (Lauderdale, 1972). The combination of progesterone, estrogens, and prostaglandin comprises the fourth phase of development. Through ultrasonography, Sirois and Fortune (1988) discovered the bovine estrous cycle is comprised of distinct wave-like patterns of follicular growth occurring anywhere from 6 to 15 d apart. Once this was discovered the goal was to find a synchronization method that allowed control of both the follicular waves and the luteal phase, therefore the GnRH-PG phase was initiated. The GnRH-PG protocols worked effectively in increasing synchronization rate in beef cattle compared to methods in the previous phases (Twagiramungu et al., 1992a; Twagiramungu et al., 1992b). Administering GnRH initiates a new follicular wave 2 to 3 d following administration, the luteal tissue that forms following GnRH injection is capable of lutetolysis via PG injection 6 to 7 d later (Twagiramungu et al., 1995). A percentage of cattle were observed coming into estrus between injections of GnRH and PG, therefore stimulating interest in the sixth phase of estrus synchronization, Progestogen-GnRH-PG.

23 16 Melengestrol Acetate Melengestrol acetate (MGA) is an orally active synthetic progestin developed in 1962 (Patterson et al., 1989). This progestin has greater hormone activity with 125 times more effective than progesterone (Lauderdale et al., 1983) and MGA binds the bovine progesterone receptor with 5.3 times greater affinity than its physiological ligand. It is used to synchronize estrus in heifers, as it is only FDA approved for use in heifers. Melengestrol acetate must be consumed by heifers at a rate of.5mg/hd/d in order for MGA to suppress estrus and inhibit ovulation (Imwalle et al., 2002). Feedlots commonly use MGA to suppress ovulation, which causes increased weight gain. The feeding level of MGA is critical for the success of an estrus synchronization protocol. Melengestrol acetate can be delivered with grain, a protein carrier, top dressed on feed, or match mixed with a larger quantity of feed. If the animals do not receive the required amount they will prematurely exhibit estrus during the feeding period. Advantages to using MGA are ease of administration, lower cost compared to other estrus synchronization products, and its potential to induce estrus in prepubertal heifers. Many estrus synchronization protocols utilize MGA; it can be used alone with natural service, in combination with GnRH and/or PG with the implementation of heat detection and AI or fixed-time AI (FTAI). This review will focus on the MGA-PG protocol in heifers. In this protocol, heifers are fed MGA for 14 d, given an injection of PG 19 d after MGA withdrawal and fixed-time AI 72 h following PG. At AI heifers are given an injection of GnRH (Figure 4).

24 17 Figure 4. Melengestrol acetate-prostaglandin and timed artificial insemination (MGA-PG & TAI) protocol for beef heifers. MGA is administered orally at.5mg/hd/d for 14 d, 19 d after MGA withdraw an injection of prostaglandin F 2α (PG) is administered, 72 h following the PG injection heifers are given an injection of gonadotropin releasing hormone (GnRH) at time AI. (Adapted from Beef Reproductive Task Force) This combination of feeding MGA for 14 d and waiting 19 d before PG injection has been developed after multiple phases of synchronization development (Figure 5). However, there was little benefit or sometimes a reduction in fertility, which could partly be contributed to the day of the estrous cycle for each heifer at treatment initiation (Figure 6). Heifers will exhibit estrus following the termination of feeding MGA, however fertility is reduced on this estrus. Low dose, high frequency pulses of LH can cause large persistent follicles to form on the ovary due to low progesterone supplementation (MGA). Follicle fertility is compromised due to altered hormone concentration and age of the follicle (Inskeep, 2004; Figure 7).

25 18 Figure 5. Development of estrous cycle control utilizing melengestrol acetate (MGA). A summary table of the 4 phases of MGA estrous synchronization protocol development. (Adapted from Patterson et al., 1989) Figure 6. Conception rates of heifers treated with Syncro-Mate-B or MGA-PG on different days of the estrous cycle. (Adapted from Patterson et al., 1989)

26 19 Figure 7. Effect of high (normal) and low dose (MGA) progesterone on LH frequency, occurrence of persistent follicles, estrogen exposure, oocyte quality, risk of early embryonic mortality and resulting fertility. (Adapted from Inskeep, 2004) When utilizing the MGA-PG estrus synchronization protocol, heifers can be estrus detected with AI carried out 12 h later and heifers that do not show signs of estrus are subjected to timed AI, with an injection of GnRH given at AI or all heifers subjected to strict timed AI with an injection of GnRH. Larson et al. (1996) conducted 2 experiments to determine conception rates of heifers to time AI after MGA-PG, however this protocol was 17 d between MGA withdraw and PG injection. In the first experiment, heifers were subjected to timed AI at 72 h after PG regardless of estrus behavior, there was a tendency for this to increase the percentage of heifers that became pregnant to AI compared with heifers inseminated 12 h after they exhibited estrus behavior. In the second experiment, the number of heifers that conceived to AI was increased by mass inseminating all heifers that did not show signs of estrus by 72 h. Lamb et al., (2000) performed a study where heifers were injected with PG on day 17 or 19 after MGA withdrawal. Heifers injected on d 19 had a shorter interval to estrus with 99% exhibiting estrus and inseminated by 72 h, compared with 74% in the 17 day after withdrawal injection group. In a study done by Johnson and Day (2004), they

27 20 compared MGA-PG (19 day PG injection) protocol with estrus detection and AI (EA), time AI (TAI) only, and estrus detection AI and clean up AI (EAC). In this study, EA resulted in 63% pregnancy rate, TAI at 60 h after PG resulted in 46.6% pregnancy, and EAC with clean up AI at 80 h after PG resulted in 63.5%. Although it has been shown MGA-PG works well for inducing puberty, synchronizing estrus, and resulting in acceptable pregnancy rates, there is another source of progesterone of interest to producers, the controlled internal drug release (CIDR). Controlled Internal Drug Release Another method of estrus synchronization in cattle is the use of a CIDR. The CIDR is a T shaped device inserted in the vagina of a cow or heifer and contains 1.38g of progesterone. This device can be somewhat costly to beef producers when compared with MGA, however CIDRs eliminate the need for cattle to be in a feedlot and/or provide bunk space as needed with MGA. Additionally, with MGA it is essential all females to be synchronized orally intake 0.5mg/hd/d for MGA to work properly. With the CIDR, there is a constant release of progesterone and no need to worry if cattle are receiving the proper amount. There is the chance a CIDR will come out of the vagina before the end of the treatment period; however, the retention rate has been about 97%, but this depends on each operation (Lamb and Larson, 2004). There are many estrus synchronization protocols utilizing CIDRs and fixed time AI that produce consistent pregnancy rates in mature cows, particularly the Co-Synch + CIDR protocol. The Co-Synch + CIDR protocol starts with an injection of GnRH and CIDR placement on day 0, CIDR removal and a PG injection on d 7, followed by AI and

28 21 a GnRH injection 54 h later (Figure 8). In cows, this protocol can yield FTAI pregnancy rates between 50 to 60 %. When utilizing the Co-Synch + CIDR protocol in heifers, Busch et al. (2007) found greater FTAI pregnancy rates were achieved when utilizing the CIDR Select protocol versus the Co-Synch + CIDR protocol (63 vs. 43%, respectively). The CIDR Select protocol requires a longer duration of progesterone administration, with the CIDR being in the vagina of the heifer for 14 d compared to 7 d. Figure 8. Co-Synch + CIDR and CIDR Select estrous synchronization protocols. The Co- Synch + CIDR starts with an injection of gonadotropin releasing hormone (GnRH) on d 0, along with the controlled internal drug release (CIDR) insert, on d 7 the CIDR is removed and an injection of prostaglandin F 2α (PG) is administered, AI occurs 54 h later with an injection of GnRH. The CIDR Select protocol starts with a CIDR insert on d 0 and is removed on d 14, 9 d following CIDR removal an injection of GnRH is administered, 7 d following an injection of PG is administered, and 72 h later GnRH is administered at AI. (Adapted from Busch et al., 2007) Additionally in the study by Busch et al. (2007), the CIDR select protocol demonstrated greater and a more synchronized estrus response prior to FTAI and the estrus following. Leitman et al. (2009) compared 4 estrus synchronization protocols utilizing a CIDR for 14 d, with or without GnRH. All treatments received an injection of

29 22 PG on d 30, heat detected, and AI approximately 12 h later. There were no differences in estrous response among treatments, conception rates to AI were consistently 60%, and final pregnancy rates were similar (83 to 92%). Kojima et al. (2004) compared MGA-Select and CIDR-Select protocols to evaluate reproductive efficacy. Both protocols utilize MGA or CIDR for 14 d, an injection of GnRH 12 d after MGA withdraw, 9 d after CIDR removal and PG injected 7 d following GnRH injection. In this study, estrus synchronization response did not differ, however AI pregnancy rate was greater (63 v. 47%, CIDR v. MGA, respectively) for heifers synchronized with the CIDR-Select protocol. By the end of the study similar pregnancy rates were observed between estrus synchronization protocols. Conclusions Current literature provides us great insight on how to influence reproduction utilizing estrous synchronization and AI, as well as manage nutrition to develop replacement heifers that are fertile prior to the first breeding season. However, many of the industry guidelines used in management practices today, are based on study results performed several decades ago. The beef industry has been changing rapidly due to advances in genetic technologies (EPDs and genomics), the increase in composite cow herds, and the changes in cattle feeding due to increased feedstuff prices driving producers to look for different options. Today s cow herds are different from cow herds a couple decades ago and it is important to continue to research important topics related to reproductive development as changes occur.

30 23 Therefore, the following 3 research chapters will address fertility in beef heifers. Chapter II will address the industry standard of developing heifers to have at least 2 estrous cycles prior to the first breeding season. This standard was established mainly by two studies from 1971 and Chapter III will evaluate the effect of BW gain at different time periods during development on puberty and pregnancy rates. From the literature, it is known BW gain plays an important role in puberty attainment and pregnancy establishment. There is no clear evidence if BW gain during specific time points, throughout development, are critical for reproductive success. And finally, Chapter IV will evaluate the reproductive efficacy of 2 estrus synchronization protocols commonly used in beef heifers. The research is limited to these 2 protocols when utilizing timed AI, which is important in reducing labor needs when implementing estrus synchronization and AI. Objectives Determine if the fertility of the first estrus differs from the fertility of subsequent estrus. Evaluate the effect of ADG at different development periods on the onset of puberty prior to the breeding season and pregnancy rates. Compare the reproductive efficacy and economics of 2, 14-d progestin estrus synchronization protocols in beef heifers.

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33 26 Lamb, G., D. Nix, J. Stevenson, and L. Corah Prolonging the MGA-prostaglandin F 2α interval from 17 to 19 days in an estrus synchronization system for heifers. Theriogenology. 53(3): Lamond, D. R Synchronization of ovarian cycles in sheep and cattle. Animal Breeding. 32(Suppl. 1): 246. (Abstr.) Larson, R., L. Corah, and C. Peters Synchronization of estrus in yearling beef heifers with the melengestrol acetate/prostaglandin F 2α system: Efficiency of timed insemination 72 hours after prostaglandin treatment. Theriogenology. 45(4): Lauderdale, J. W Effect of corticoid administration in bovine pregnancy. J. Am. Vet. Med. Assoc. 160(6): Leitman, N., D. Busch, D. Mallory, D. Wilson, M. Ellersieck, M. Smith et al Comparison of long-term CIDR-based protocols to synchronize estrus in beef heifers. Anim. Reprod. Sci. 114(4): Lesmeister, J., P. Burfening, and R. Blackwell Date of first calving in beef cows and subsequent calf production. J. Anim. Sci. 36(1): 1-6. Lynch, J. M., G. C. Lamb, B. L. Miller, R. T. Brandt Jr, R. C. Cochran, and J. E. Minton Influence of timing of gain on growth and reproductive performance of beef replacement heifers. J. Anim. Sci. 75(7): Nilsson, E. E., and M. K. Skinner Progesterone regulation of primordial follicle assembly in bovine fetal ovaries. Mol. Cell. Endocrinol. 313(1-2): Patterson, D. J., G. H. Kiracofe, J. S. Stevenson, and L. R. Corah Control of the bovine estrous cycle with melengestrol acetate (MGA): A review. J. Anim. Sci. 67(8): Patterson, D. J., R. C. Perry, G. H. Kiracofe, R. A. Bellows, R. B. Staigmiller, and L. R. Corah Management considerations in heifer development and puberty. J. Anim. Sci. 70(12): Rahe, C. H., R. E. Owens, J. L. Fleeger, H. J. Newton, and P. G. Harms Pattern of plasma luteinizing hormone in the cyclic cow: Dependence upon the period of the cycle. Endocrinology. 107(2): Roberts, A. J., T. W. Geary, E. E. Grings, R. C. Waterman, and M. D. MacNeil Reproductive performance of heifers offered ad libitum or restricted access to feed for a one hundred forty-day period after weaning. J. Anim. Sci. 87(9):

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35 28 CHAPTER II: Effect of pubertal status and number of estrous cycles prior to the breeding season on pregnancy rate in beef heifers R. A. Vraspir 1*, A. F. Summers 1, A. J. Roberts 2, and R. N. Funston 1 1 University of Nebraska, West Central Research and Extension Center, North Platte 69101; 2 USDA-ARS, Fort Keogh Livestock and Range Research Laboratory, Miles City, MT Abstract Three experiments were conducted to evaluate whether pubertal status prior to breeding influences pregnancy rate in beef heifers. Records were collected at West Central Research and Extension Center, North Platte, Neb., from 2002 to 2011 (Exp. 1; n = 1,005) and Gudmundsen Sandhills Laboratory, Whitman, Neb., from 1997 to 2011 (Exp. 2; n = 1,253). Heifers in Exp. 1 and 2 were classified as either being pubertal or non-pubertal at the start of breeding. In Exp. 3, (n = 156) heifers were classified by number of estrous cycles (0, 1, 2, 3, or 4) exhibited prior to breeding. In Exp. 1 and 2, pubertal heifers were heavier (P 0.04) and older (P < 0.07) at start of breeding and had a greater (P < 0.01) overall pregnancy rate (94 vs. 88 ± 2%, 90 vs. 82 ± 2% in Exp. 1 and 2, respectively) than non-pubertal heifers. Pubertal heifers also tended (P = 0.08) to have greater AI pregnancy rate (62 vs. 56 ± 4%, Exp. 1), produced more calves within the first 21 d of calving (P < 0.01), and weaned older (P = 0.05), heavier (P < 0.01) calves than heifers that had not reached puberty (Exp. 2). In Exp. 3, heifers pubertal prior to the breeding season had greater (85 ± 8%, P = 0.05) pregnancy rates (68 ± 8%) than nonpubertal heifers and pregnancy rate tended (P = 0.15) to be influenced by the number of